JOURNAL

Contents

Original Papers

Highly efficient method of Lithospermum erythrorhizon transformation using domestic Rhizobium rhizogenes strain A13

Kanade Tatsumi, Takuji Ichino, Noboru Onishi, Koichiro Shimomura, Kazufumi Yazaki . . . . . . . 39 [PDF] [Supplement1] [Supplement2]

Lithospermum erythrorhizon, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact L. erythrorhizon plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in L. erythrorhizon cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for L. erythrorhizon transformation using hairy roots by Rhizobium rhizogenes strain A13, present domestically in Japan. Hairy roots of L. erythrorhizon were generated from explants of the axenic shoots that were infected with R. rhizogenes strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50–70%. Although methods for the systematic stable transformation of L. erythrorhizon plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of L. erythrorhizon genes.

A novel FLOURY ENDOSPERM2 (FLO2)-interacting protein, is involved in maintaining fertility and seed quality in rice

Rintaro Suzuki, Tomohiro Imamura, Yoko Nonaga, Hiroaki Kusano, Hiroshi Teramura, Ken-Taro Sekine, Tetsuro Yamashita, Hiroaki Shimada . . . . . . . 47 [PDF] [Supplement1]

Crop plants accumulate a large amount of storage starch and storage proteins in the endosperm. Genes involved in the biosynthesis of these substances work in concert during development of the rice endosperm. The rice flo2 mutant produces aberrant seeds with reduced grain quality. FLOURRY ENDOSPERM 2 (FLO2), the causative gene of the flo2 mutant, is considered to be a regulatory protein that controls the biosynthesis of seed storage substances. FLO2 contains tetratricopeptide repeat (TPR) motifs that may mediate protein–protein interactions. In this study, we identified the protein that interacts with the TPR motif of FLO2. We generated a transformant that produced the FLAG-tagged fusion FLO2 protein in the flo2 mutant and used this in the shotgun proteomic analysis. A protein, which we named FLOC1, interacted with FLO2. In vitro pull-down assays indicated that the TPR motif was involved in this interaction. A knock-down transformant of FLOC1 showed significantly reducted fertility and generation of seeds with abnormal features. These findings suggest that FLOC1 is involved not only in seed fertility but also in seed quality. These phenotypes were also observed on the RNAi transformants of the flo2 mutant although the effect of the flo2 mutation remained. these findings imply that there is a difference in the functions of FLO2 and FLOC1 although both of appear to be involved in the control of seed quality during seed formation.

Effect of circularly polarized light on germination, hypocotyl elongation and biomass production of arabidopsis and lettuce: Involvement of phytochrome B

Enkhsukh Lkhamkhuu, Kazunori Zikihara, Hitomi Katsura, Satoru Tokutomi, Takafumi Hosokawa, Yoshihisa Usami, Mitsuyoshi Ichihashi, Junji Yamaguchi, Kenji Monde . . . . . . . 57 [PDF] [Supplement1]

Circular dichroism (CD), defined as the differential absorption of left- and right-handed circularly polarized light (CPL), is a useful spectroscopic technique for structural studies of biological systems composed of chiral molecules. The present study evaluated the effects of CPL on germination, hypocotyl elongation and biomass production of Arabidopsis and lettuce. Higher germination rates were observed when Arabidopsis and lettuce seedlings were irradiated with red right-handed CPL (R-CPL) than with red left-handed CPL (L-CPL). Hypocotyl elongation was effectively inhibited when Arabidopsis and lettuce seedlings were irradiated with red R-CPL than with red L-CPL. This difference was not observed when a phytochrome B (phyB) deficient mutant of Arabidopsis was irradiated, suggesting that inhibition of elongation by red R-CPL was mediated by phyB. White R-CPL induced greater biomass production by adult Arabidopsis plants, as determined by their fresh shoot weight, than white L-CPL. To determine the molecular basis of these CPL effects, CD spectra and the effect of CPL on the photoreaction of a sensory module of Arabidopsis phyB were measured. The red light-absorbing form of phyB showed a negative CD in the red light-absorbing region, consistent with the results of germination, inhibition of hypocotyl elongation and biomass production. L-CPL and R-CPL, however, did not differ in their ability to induce the interconversion of the red light-absorbing and far-red light-absorbing forms of phyB. These findings suggest that these CPL effects involve phyB, along with other photoreceptors and the photosynthetic process.

Inactivation of retrotransposon Tos17^Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing

Yanchang Luo, Dongsheng Tian, Joanne Chin Yi Teo, Kar Hui Ong, Zhongchao Yin . . . . . . . 69 [PDF]

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17^Chr.7) and 10 (Tos17^Chr.10), respectively. Previous studies demonstrated that only Tos17^Chr.7 is active in transposition during tissue culture. Tos17^Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17^Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17^Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17^Chr.7 in D873, designated as Tos17^D873, has an 873-bp DNA deletion in the pol gene of Tos17^Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17^D873 was activated in D873 calli, no transposition of Tos17^D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17^Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17^Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17^D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.

Short Communications

Development of the sequence-characterized amplified region (SCAR) marker for distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries

Takahiro Muto, Takanori Kuronuma, Masaya Ando, Hiroyuki Katsuoka, Zentaro Inaba, Hitoshi Watanabe . . . . . . . 77 [PDF]

Distinction of intergeneric hybrids between Argyranthemum frutescens (L.) Sch. Bip. and Rhodanthemum gayanum (Cross. & Durieu) B.H. Wilcox, K. Bremer & Humphries is important for the development of new intergeneric hybrids. The aim of this study was to develop a simple and cost-effective DNA marker to distinguish between a large number of putative hybrids of A. frutescens × R. gayanum during primary selection. We designed sequence-characterized amplified region (SCAR) markers based on the genetic sequences of the internal transcribed spacer (ITS) region for A. frutescens and R. gayanum. Specific SCAR markers for A. frutescens amplified bands in A. frutescens and hybrids but not in R. gayanum. In contrast, specific SCAR markers for R. gayanum amplified bands in R. gayanum and hybrids but not in A. frutescens. Additionally, we confirmed the reproducibility of the SCAR marker using 31 cultivars. The findings demonstrated that these SCAR markers could be used to distinguish putative hybrids between A. frutescens and R. gayanum. This is the first report of the development of a SCAR marker to facilitate distinction of these hybrids.

Application of cucumber mosaic virus to efficient induction and long-term maintenance of virus-induced gene silencing in spinach

Hangil Kim, Yasuyuki Onodera, Chikara Masuta . . . . . . . 83 [PDF] [Supplement1]

Virus-induced gene silencing (VIGS) is a useful tool for functional genomics in plants. In this study, we tried to apply cucumber mosaic virus (CMV) to efficient induction of VIGS in spinach. Although VIGS for spinach had been previously developed based on two viruses (beet curly top virus and tobacco rattle virus), they still have some problems with systemic movement and long-term maintenance of VIGS in spinach. Although ordinary CMV strains infect spinach inducing distinct mosaic symptoms, using a CMV pseudorecombinant, we can modify the viral pathogenicity to attenuate viral symptoms that may mask the silencing phenotype. We here successfully demonstrated the viral ability to silence the phytoene desaturase (PDS) and the dihydroflavonol 4-reductase (DFR) genes in spinach. Because CMV could quickly induce VIGS even at 7–10 days postinoculation and the virus did not disappear even at the flowering stage, this CMV-based VIGS system would contribute to functional genomics in spinach and especially to the elucidation of molecular mechanisms for some properties unique to spinach such as plasticity of sex expression; the CMV-induced VIGS can last until the flowering stage after the virus was inoculated onto the seedling.

Notes

Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system

Sakihito Kitajima, Kenji Miura, Junko Yasuda . . . . . . . 89 [PDF]

Agroinfiltration, the infiltration of plants with Agrobacterium harboring a plasmid that contains a specific gene, is used to transiently express a gene in a heterologous organism. Using the “Tsukuba system”, greater amounts of target protein accumulate compared with usual expression plasmids. Reported host plants, including Nicotiana benthamiana, a common plant for agroinfiltration, need several weeks after sowing to grow enough for infection. To shorten the culture period and, thereby, improve target protein production, we tested sprouts as host plants. Sprouts were grown in the dark to encourage elongation so that vacuum infiltration becomes easier, and this was followed by a few days of exposure to illumination before infection with pBYR2HS-EGFP, the EGFP expression plasmid of the Tsukuba system. Among six tested species of Fabaceae and Brassicaceae, radish showed the highest transient expression. Among six tested radish cultivars, Kaiware, Hakata, and Banryoku provided the best results. Culturing for 5 day, including 1 day of imbibition and 1 to 2 day of exposure to illumination resulted in EGFP fluorescence in 80% of the cotyledon area. Thus, a remarkable amount of EGFP was obtained only 8 day after seed imbibition. The EGFP amount in Kaiware cotyledons was comparable with Rubisco at ∼0.7 mg/g fresh weight. Kaiware sold in supermarkets could also be used, but resulted in lower expression levels.

Inhibitory effects of Alpinia zerumbet extract against plant virus infection in solanaceous plants

Mari Narusaka, Yasuyuki Yamaji, Misugi Uraji, Tadashi Hatanaka, Yoshihiro Narusaka . . . . . . . 93 [PDF]

Tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) are critical pathogens causing severe crop production losses of solanaceous plants. The present study was undertaken to evaluate the antiviral effects of extracts of Alpinia plants on ToMV and TMV infection in Nicotiana benthamiana. The aqueous extracts of Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith and Alpinia kumatake, which grow widely in subtropical and tropical regions including East Asia, were effective in reducing ToMV infection when plants were treated prior to virus inoculation. We also found that the extracts of A. zerumbet isolated from Okinawa (Japan), locally referred to as shima-gettou, strongly inhibited ToMV and TMV infection. To obtain an active fraction, the aqueous extract of A. zerumbet isolate OG1 was separated by ethyl acetate, and the antiviral active compound was found to be present in the water layer. Based on our results, the extract of Alpinia plants has potential as an antiviral reagent for practical application in solanaceous crop production.

Diversity of the bacteria in the roots of sugarcane used to produce Wasanbon in Kagawa, Japan

Supriadi, Natsuki Shimoura, Masahiro Miyaji, Shigeyuki Tajima, Mika Nomura . . . . . . . 99 [PDF] [Supplement1]

Soil bacteria are an important factor in the cycle of nutrients in soil, while root bacteria in the internal tissues of plants can promote plant growth. The aim of this research was to study the diversity of root bacteria of sugarcane grown in Kagawa, Japan. The sugarcane, derived from Saccharum sinense and grown only in this area, is used as a raw material for Wasanbon (a fine-grained Japanese sugar) and is characterized by thin stalks and a low sugar content. To determine its bacterial diversity, bacterial DNA was extracted from the soil and roots, and 16S rRNA genes were sequenced. A total of 1259 operational taxonomic units (OTUs) were detected in the root bacteria and 3894 OTUs in the soil bacteria. The α-diversity between the soil and root bacteria was significantly different. The most abundant class of root and soil bacteria was proteobacteria at more than 50 and 30%, respectively. The endophyte bacteria of rhizobium were also isolated and the nifH gene was detected. The relationship between the application of nitrogen-fixing bacteria and the production of Wasanbon should be studied.

Image analysis of stress-induced lignin deposition in Arabidopsis thaliana using the macro program LigninJ for ImageJ software

Masato Nakamura, Tomoaki Kamehama, Yasushi Sato . . . . . . . 105 [PDF] [Supplement1]

In vascular plants, lignin is deposited during morphogenesis but also under stress conditions. Assessing the degree of stress-induced lignin deposition is complicated because it occurs locally and irregularly in plant tissues. In this study, we developed a macro program, LigninJ, for the open-source software ImageJ to automatically and efficiently determine areas and levels of lignification after Wiesner (phloroglucinol-HCl) staining. We used the CIELAB color space for detection of red color following the Wiesner reaction. In addition, LigninJ has a function for adjusting the background level and its white balance to reduce biases that are inherent to individual color images. Furthermore, LigninJ can be used for batch analyses of multiple images, taking about 2 s per image. In this study, we analyzed wound-induced lignin deposition in cotyledons of the Arabidopsis thaliana ecotypes Landsberg erecta and Columbia and assessed ectopic lignin depositions in roots of lignescence (lig) mutants of Arabidopsis. Our results confirmed that this method is efficient for evaluating the degree of stress-induced lignin deposition.

Identification of new pentatricopeptide repeat proteins, MREF1 and 2, involved in mitochondrial RNA editing, using computational target RNA prediction

Takehito Kobayashi, Yusuke Yagi, Taizo Tamura, Takahiro Nakamura . . . . . . . 111 [PDF] [Supplement1]

C-to-U RNA editing has been widely observed in mitochondrial and plastid RNAs in plants. The editing sites are known to be recognized by pentatricopeptide repeat (PPR) proteins, which belong to one of the largest protein families in vascular plants. PPR proteins are sequence-specific RNA-binding proteins that participate in various steps of organelle RNA metabolism, such as cleavage, stabilization, splicing, translation, and editing. Elucidating the underlying mechanisms of sequence-specific RNA recognition by PPR proteins expanded our understanding of the role of PPR proteins in plant organellar RNA editing and enabled the computational prediction of target RNA-editing sites for PPR proteins of interest. Combining computational prediction and experimental verification, we identified three new PPR proteins involved in mitochondrial RNA editing: At1g56570, known as PGN for RNA editing of nad6_leader_-73 and cox2_742, At4g04370 for RNA editing of nad5_242, and At2g41080 for atp1_1292. Therefore, At4g04370 and At2g41080 were designated as mitochondrial RNA-editing factor 1 (MREF1) and MREF2, respectively. This study supports the use of computational prediction in establishing connections between PPR proteins and specific RNA-editing sites, which are important for maintaining various physiological processes, such as plant development, embryogenesis, and biotic- and abiotic-stress responses.